International Journal of Agricultural Sciences ISSN: 2167-0447 Vol. 2 (6), pp.198-203, June, 2012. Available online at www.internationalscholarsjournals.org © International Scholars Journals

Full Length Research Paper

Variation in resistance to coffee berry disease ( kahawae) among germplasm progenitors at coffee research institute (tacri)

D.J.I. Mtenga1* and S.O.W.M. Reuben2

1Tanzania Coffee Research Institute, P.O. Box 3004, Moshi, Tanzania.

2Sokoine University of Agriculture, Department of Crop Science and Production, P.O. Box 3005, Morogoro, Tanzania.

Received February 23, 2012; Accepted June 03, 2012

Four trials were executed at the Tanzania Coffee Research Institute (TaCRI) from September 2006 to April 2007 and at the Coffee Rust Research Center (CIFC), Portugal from March 2007 to June 2007 to evaluate variation in resistance to Coffee Berry Disease (CBD) caused by Colletotrichum kahawae within and among four arabica varieties. The varieties tested were Bourbon, Compacts (Catimor), Rume sudan and Hybrid de Timor obtained from TaCRI germplasm. Local CBD isolate was used in the trials at TaCRI while at CIFC Portugal, four CBD isolates T3, Ca1, Z9 and Q2 were used in the resistance evaluation. There was significant (P ≤ 0.05) variation against CBD within and among varieties against most isolates. Accessions PNI086, VCE1589 and VC299 in varieties Compacts, Hybrid de Timor and Rume sudan respectively showed high resistance while variety Bourbon a control, showed high levels of susceptibility. It is therefore necessary in breeding programmes to select the best accessions within varieties for development of superior coffee varieties with resistance to CBD.

Key words: Coffee berry disease; breeding; ; resistance sources

INTRODUCTION

Coffee (Coffea) is one of the most important beverages in Coffea arabica L. and Coffea canephora Pierre ex the world with a current estimated value of 10 billion US$ Froehner. In the world coffee trade, Arabica coffee and it is ranked second largest trade commodity next to accounts for about 70% while Robusta coffee contributes petroleum (Luiz et al., 2005). Coffee is also an extremely about 30% (ICO, 2006). Coffee is Tanzania’s largest important source of national export revenue and internal export crop. It contributes approximately $115 million to cash income for farmers in many poor countries from export earning, and provides employment to some Africa, Asia and Latin America (ICO, 2005). The two most 400,000 families. About 95 percent of coffee is grown by economically important species of the genus Coffea are smallholders on average holdings of 1–2 hectares, and 5 percent is grown on estates. Almost all of Tanzania’s coffee production is exported (Baffes, 2003). ______Numerous production constraints have been mentioned. However, the most serious one is diseases, mainly coffee *Corresponding Author’s E-mail: [email protected] berry disease (CBD) (Colletotrichum kahawae, Waller

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Table 1: Varieties and accessions studied

Variety Accessions Rume sudan VC 298, VC 299, VC 506, VC509, and VC510

Hybrid de Timor RRC70, RRC72, VCE 1594, VCE 1587and VCE1589 Catimor/Compacts PNI 088, PNI 086, PNI 087, CR 124 and CR127

Bourbon N5, N39, N100, N197 and N218

Note: Bourbon is a commercial (susceptible) variety, used as a check.

and Bridge) and coffee leaf rust (CLR) (Hemileia Four Coffee Berry Disease isolates from Tanzania (T3), vastatrix, Berk et Br). Coffee Berry Disease is an Kenya (Q2), (Z9) and (Ca1) were anthracnose of the green and ripening berries while used for the experiments done at CIFC- Portugal while a Coffee Leaf Rust is a fungal disease which attacks leaves local isolate extracted from freshly infected berries was of both Arabica and Canephora coffee (Wrigley, 1988). used for the experiment done in Tanzania. Together, the two diseases viz CBD and CLR control account for 40% of total production cost for small scale Experimental procedure coffee producers in the country (Teri et al., 2004; Mtenga et al., 2006).

The Arabica coffee improvement programme at the Tanzania Coffee Research Institute (TaCRI) has Fully matured, red-ripe coffee berries were handpicked achieved a considerable success in developing CBD and from the trees from centre rows of each accession CLR resistant varieties. However some inconsistencies involved from the germplasm plot. These were pulped on the levels of resistance have been observed in some using a small hand pulper in separate lots for each of the progenitors used in the development of the accession. The pulper hopper was washed thoroughly varieties (Walyaro, 2004). Establishment of levels of using clean tap water under high pressure to ensure all resistance in the progenitors of resistance will lead to the cherries from previous lot is cleaned out of the pulper identification of superior which will be used for before pulping the next lot. This procedure was followed development of improved varieties with consistent, stable for all lots. Pulped cherries from each accession were put and durable resistance to the diseases. The objective of in separate containers, placed under room temperature the current study was to evaluate the resistance levels for 72 hours for fermentation. Then the cherries were within and among the main progenitors of CBD washed and dried under shed separately in partitioned resistance used at TaCRI. wire-mesh trays for one week before they were pre- germinated to raise hypocotyls.

Sources of inoculums MATERIALS AND METHODS

According to Cook (1973b) green infected berries with Varieties and accessions studied active lesions are the best source of inoculum for initial isolation because of low contamination with other non The materials which were used in this work are three pathogenic Colletotrichum spp. and optimum coffee varieties from Tanzania Coffee Research Institute pathogenicity of the isolates. Initial isolation of the CBD germplasm which are the main progenitors for CBD pathogen was achieved by incubating green infected resistance. In each variety, five accessions were berries from unsprayed susceptible variety Bourbon evaluated as shown in Table 1. (N39) at 200C-240C on moist sterilized sand in closed but somewhat ventilated plastic boxes for 10 days (Cook, CBD isolates tested 1973b). This was followed by planting the conidia on

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Table 2: Variation in resistance to coffee berry disease (CBD) 0-4 score within varieties against Q2, Ca1, T3, Z9 CBD isolates at CIFC Portugal and Local one in Tanzania Varieties Accessions Isolates Mean Scores Rume sudan Q2 Ca1 T3 Z9 Local VC 298 0.350 2.550 1.000 0.733 0.160 VC 299 0.817 1.133 1.067 0.500 0.040 VC 506 0.683 2.233 0.900 0.983 0.040 VC 509 0.767 3.900 1.217 0.833 0.060 VC 510 0.633 3.650 0.850 0.750 0.060 mean 0.650 2.693 1.007 0.760 0.072 CV (%) 29.620 8.790 32.950 20.650 89.580 SE± 0.000 0.137 0.000 0.091 0.000

LSD0.05 ns 0.446 ns 0.298 ns Hybrid de Timor VCE 1587 0.883 2.583 1.967 1.833 0.010 VCE 1589 0.783 2.400 0.633 0.950 0.050 VCE 1594 3.717 3.883 1.933 2.183 0.340 RRC 70 2.283 3.933 1.417 2.183 0.440 RRC 72 1.300 3.617 2.150 1.600 0.050 mean 1.793 3.283 1.620 1.750 0.178 CV (%) 15.260 6.360 19.500 18.980 70.300 SE± 0.158 0.121 0.183 0.192 0.061

LSD0.05 0.516 0.395 0.595 0.625 0.189 Compacts PNI 086 0.567 1.850 0.950 0.717 0.000 PNI 087 2.033 3.800 1.600 1.783 0.010 PNI 088 0.050 3.85 0.167 0.283 0.000 CR 124 1.350 3.900 2.317 2.367 0.470 CR 127 0.850 3.967 1.817 0.750 0.040 mean 0.970 3.473 1.370 1.180 0.104 CV (%) 23.030 5.590 12.830 20.140 35.630 SE± 0.129 0.113 0.102 0.153 0.158

LSD0.05 0.421 0.367 0.332 0.498 0.487 Bourbon N 5 4.000 4.000 4.000 4.000 3.590 N 39 4.000 4.000 4.000 4.000 3.510 N 100 4.000 4.000 4.000 4.000 1.980 N 197 4.000 4.000 4.000 4.000 1.430 N 218 4.000 4.000 4.000 4.000 3.390 mean 4.000 4.000 4.000 4.000 2.780 CV (%) 0.000 0.000 0.000 0.000 14.620 SE± 0.000 0.000 0.000 0.000 0.211

LSD0.05 ns ns ns ns 0.650

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3.4% malt extract agar containing 0.04% streptomycin. A RESULTS 6 spore suspension containing 2 x10 spores/ml was prepared from these pure cultures. The suspension was There was significant (P≤0.05) variation in resistance to tested and proved spore viability in excess of 80%.This coffee berry disease (CBD) within varieties Hybrid de procedure was followed in extraction and preparation of Timor, Compacts and Bourbon against a local isolate at inoculum of the local CBD isolate used in the experiment TaCRI Tanzania (Table 2). No significant variation was conducted in Tanzania. For the experiments carried out observed between accessions in Rume sudan variety. in Portugal; isolates were maintained in malt extract agar Rume sudan, Hybrid de Timor and Compacts showed (MEA) and preserved for long periods in agar slants. high levels of resistance with mean scores of 0.072, 0.178 and 0.104 respectively. The control variety Test for resistance Bourbon showed susceptibility with mean score of 2.780. At CIFC Portugal when coffee berry disease isolate T3 One hundred seeds of each variety were sown out, with was used, significant (P≤ 0.05) variation in resistance the parchment removed, in moist sterilized media of sand was observed within varieties in two varieties of Hybrid and top forest soil placed in plastic boxes with closely de Timor and Compacts while the variety Rume sudan fitting transparent lids, keeping the boxes at normal room and Bourbon did not show significant variation in temperatures (20-240C). The seedlings were ready for resistance within the varieties (Table 2). inoculation when they had hypocotyl stem of 3-5cm long, High level of resistance was observed in varieties Rume usually within 5-6 weeks after sowing out the seeds. At sudan, Hybrid de Timor and Compacts with mean scores this stage the cotyledons are still enclosed in the testa. 1.007, 1.620 and 1.370 respectively. However, some Just before inoculation the lid was removed from the accessions within varieties Hybrid de Timor (RRC 72) plastic box, the seedlings sprayed with the standard CBD and Compacts (CR 124) had exceptional high inoculum (2 x 106 spores/ml) by means of a small susceptibility scores of 2.150 and 2.317 respectively. atomizer and the box was immediately closed again. A Bourbon variety showed highest susceptibility with mean repeat inoculation was applied after 48 hours. The score of 4.000. Accessions within varieties Rume sudan, temperature of 22-240C was maintained for four days for Hybrid de Timor and Compacts showed significant (P≤ successful infection, at the same time relative humidity in 0.05) variation in resistance to CBD isolate Ca1 (Table the boxes was maintained at 100%. This was followed by 2). All the varieties showed susceptibility to this isolate. incubation period at a lower temperature of (19-200C) Mean scores for varieties against this isolate were 2.693, with the lids removed from the boxes to allow for normal 3.283, 3.473 and 4.000 respectively for varieties Rume humidity. The first symptoms became noticeable within sudan, Hybrid de Timor, Compacts and Bourbon a one week after the first inoculation, but full expression of control variety. However, accession VC299 within variety the disease susceptibility was attained at two weeks’ Rume sudan and PNI 086 within Compacts showed good time. At the end of the incubation period seedlings were levels of resistance with scores of 1.133 and 1.850 individually scored for disease symptoms developed on respectively. Three varieties, Rume sudan, Hybrid de the hypocotyl stem using a scale with range 0-4 (Van der Timor and Compacts showed significant (P≤0.05) Graaf, 1981). variation in resistance to CBD within varieties against CBD isolate Z9 at CIFC Portugal (Table 2). There was no significant variation in resistance within variety Bourbon Data analysis which was highly susceptible to this isolate.

The varieties Rume sudan, Hybrid de Timor and A mean grade of infection obtained from the scores (G) Compacts showed resistance with mean scores of 0.760, were calculated for accessions in each replication as 1.750 and 1.180 respectively. Bourbon variety showed 4 susceptibility with mean score of 4.000. However, within G = 1/N ∑ ini Hybrid de Timor, accessions VCE 1594 (2.183), RRC 70 i=1 (2.183); Compacts accession CR 124 (2.367) showed susceptibility to the Z9 CBD isolate at CIFC, Portugal. Rume sudan, Hybrid de Timor and compacts though Where, i is the disease class, ni is the number of differed significantly showed good levels of resistance seedlings in class i and N is the total number of seedlings against this isolate while Bourbon the control was highly scored (Omondi et al., 2001). Calculated score means susceptible. Observation on the reaction of CBD isolate were subjected to analysis of variance, and where a Q2 showed that there was a significant variation (P≤0.05) significant variation was found; means were separated in resistance to this isolate within varieties in varieties using Duncan’s New Multiple Range Test. Hybrid de Timor and Compacts while there was no

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Table 3: Variation in resistance to coffee berry disease (CBD) 0-4 score among varieties against Q2, Ca1, T3, Z9 CBD isolates at CIFC Portugal and a ‘Local’ one in Tanzania

Variety Isolates Mean scores Q2 Ca1 T3 Z9 Local Rume sudan 0.650 2.693 1.007 0.760 0.072 Hybrid de Timor 1.793 3.283 1.620 1.750 0.178 Compacts 0.970 3.473 1.370 1.180 0.104 Bourbon 4.000 4.000 4.000 4.000 2.780 mean 1.853 3.362 1.999 1.922 0.784 CV (%) 41.770 20.510 29.260 31.170 66.790 SE± 0.346 0.308 0.522 0.273 0.233

LSD0.05 1.067 0.949 0.777 0.840 0.719

significant variation in resistance to this isolate in populations (Van der Vossen et al., 1976; Ribeiro et al., varieties Rume sudan and Bourbon (Table 2). 2001). These methods in order to serve the purpose must Significant (P ≤ 0.05) variation was observed among be reliable, simple, rapid and feasible to allow the varieties against a local isolate (Table 3). Three handling of large plant populations with minimal chance varieties Rume sudan, Hybrid de Timor and Compacts for escapes (FAO, 1984). did not differ in their resistance level among themselves Coffee breeding programmes are limited in progress due but showed significant difference with the control variety to the narrow genetic base of cultivated varieties Bourbon which showed susceptibility. There was especially for pest and disease resistance (Van der significant (P≤0.05) variation among varieties against the Vossen, 1985). However, nowadays natural and artificial isolate T3 (Table 3). Rume sudan, Hybrid de Timor and hybrids derived from Coffea arabica x Coffea canephora Compacts were relatively resistant to this isolate while are intensively used as source of resistance to coffee Bourbon displayed significant susceptibility to the T3 berry disease caused by Colletotrichum kahawae isolate. There was significant (P≤0.05) variation in (Lashermes et al., 1996b). A natural hybrid, Hybrid de resistance among varieties against Ca1 isolate at CIFC, Timor (Timor hybrid) and the artificial hybrid Catimor Portugal (Table 3). All varieties were susceptible with (Compacts) and other derivatives such as Sarchimor are mean scores ranging from 2.693 for Rume sudan to some of the most widely utilized materials both as 4.000 for the susceptible control Bourbon. On the overall, released varieties and breeding lines especially in central there was a significant variation (P≤0.05) in resistance to America (Silveira et al., 2003). CBD isolate Z9 among varieties (Table 3). Significant Variation in resistance to CBD within varieties in Rume (P≤0.05) variation in resistance to CBD among varieties sudan and Compacts (Catimor) have been reported when was observed against the isolate Q2 (Table 3). Varieties large populations of these varieties from diverse Rume sudan, Hybrid de Timor and Compacts showed geographic origins were worked on (Agwanda et al., high resistance to isolate Q2 with mean scores 0.650, 1997). The unique resistance observed on accessions 1.793 and 0.970 respectively. The variety Bourbon VC 299 (Rume sudan) and PNI 086 (Compacts) against showed consistently high susceptibility among varieties. Ca1 isolate which is the most aggressive of all could be that for this particular isolate, there are no gene sequences in the other accessions that give gene DISCUSSION products to confer resistance while these sequences are available in accessions VC 299 and PNI 086 in Rume sudan and Compacts respectively. The varieties Rume In order to increase efficiency, reduce time and cost in sudan, a semi wild arabica coffee variety, Hybrid de breeding programmes, breeders have developed Timor a spontaneous hybrid between C. arabica x C. screening methods in which plants as young as possible canephora and Catimor (Compacts) a hybrid derived are exposed to high concentrations of specific inoculum from Hybrid de Timor x Catuai are reputed for their in order to identify resistant plants or lines in segregating resistance to coffee berry disease. The resistance in

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Rume sudan is controlled by two major genes R.R while General levels of resistance to CBD disease in the in Hybrid de Timor it is controlled by a single major evaluated progenitors of Rume sudan, Hybrid de Timor gene T (Van der Vossen and Walyaro, 1980; Agwanda and Compacts against isolates T3, Z9, Q2 at CIFC et al., 1997; Omondi et al., 2001). Resistance in Portugal and the local isolate at TaCRI-Tanzania Catimor (Compacts) is controlled by one gene easily proved that there is good resistance against the current transferable Ck-1 identified recently (Gichuru et al., CBD isolates in Tanzania. However if varieties 2006).Variation in resistance to CBD within varieties developed in Tanzania are introduced to West Africa or can be explained basing on two possible reasons. The the isolate Ca1 from Cameroon is introduced to first possibility may be due to natural out cross in Tanzania; there is a possibility of a threat against pollination with susceptible varieties in neighbouring which all the progenitors fall susceptible when plots since even though Coffea arabica is a self fertile evaluated at CIFC-Portugal. When selecting parents for species, there is chance outcross of up to 10% (Van der particular crossing in developing new improved hybrids Vossen, 1985). The second possibility may be due to for CBD resistance, selection within varieties is segregation especially in hybrid varieties Hybrid de important. Timor and Compact (Catimor) but less so in pure lines Bourbon and Rume sudan. Thus, there is scope for further selection and genetic advancement within the ACKNOWLEDGEMENTS varieties for resistance against the CBD isolates. Many authors have found out that the aggressiveness This work was financed by the European Union and of the CBD isolate population shows variability between TaCRI stakeholder’s fund. Assistance from field staff at isolates from the same or different geographic origins TaCRI and technical staff at CIFC is highly appreciated. (Van der Vossen, 1985; Omondi et al., 2001). A study This work is published with the permission of the Chief by Bieysse et al. (unpublished data INCO- project ICA4- Executive Director, TaCRI, Tanzania. CT-2001-10008) on isolates from Cameroon, Kenya, Burundi, Tanzania, Rwanda, , and REFERENCES concluded that the strains might be classified in two groups- East Africa and Cameroon, each Agwanda CO, Lashermes P, Trouslot P, Combes MC, Charrier A geographical population showing a strong homogeneity (1997). Identification of RAPD markers for resistance to coffee between the isolates (Silva et al., 2006). 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